Novaseq 6000 s2 reagent kit

RNA sequencing and gene expression profiles were performed to determine the maturation of hiPSC-CMs in isolated culture versus BCP. Isolated total RNA was isolated from cultured hiPSC-CMs and BCP samples using a RNeasy Blood and Tissue kit (Qiagen, Carlsbad, CA, USA). To construct the sequencing library for Nova Seq 6000, 2 μg of total RNA was used for library construction with the Illumina TruSeq Stranded total RNA Library Prep Kit (cat # 20020596, CA, USA). Next, paired-end sequencing was performed using the Illumina Nova Seq 6000 sequencing instrument with NovaSeq 6000 S2 reagent kit (cat # 20012860, CA, USA), according to the manufacturer’s instructions, yielding 150-bp paired-end reads. EdgeR software was used for differential expression analysis of RNA sequencing and gene expression profiles with three biological replications. This analysis assumed a negative binomial distribution for statistics. To eliminate biological variation, the screening of differential genes needed to be evaluated in terms of difference multiples and significance levels. The screening threshold for differential genes in this analysis was set to |log2 (Fold Change) | > 1, FDR < 0.01.

Genomic DNA was prepared from the L. infantum resistant clones (1044Cl₁ and 8012Cl₂) and the wild-type (WT) parent line. DNA was quantified fluorometrically, and 2 μg of material was used for library preparation and sequencing on an Illumina NovaSeq 6000 sequencer with a NovaSeq 6000 S2 reagent kit (200 cycles). The reads obtained were aligned against the L. infantum JPCM5 reference genome (Tritryp_v9) using BWA-MEM software. Single nucleotide polymorphisms (SNPs) and indels were identified using GATK software. SNPs and indels that were present in the WT line but that were not located in coding sequences or that did not generate an amino acid alteration in the resistant lines (synonymous mutations) were not considered. To determine the influence of the mutation, the allele frequency (homozygous/heterozygous), the impact of the mutation (as a property of the altered amino acid and indel), and the presence of the mutated gene in both resistant lines were taken into account. Genes with identical mutations in both resistant lines and genes harboring several mutations (≥3) were considered less important, as they likely reflect natural polymorphisms. Additionally, the copy number variations (CNVs) in each chromosome were determined by calculating the ratio of the number of normalized reads from the resistant line compared to the number from the susceptible parent line.